Antibodies specific for the carbohydrate antigen GaIalpha1-3Galbeta1-4GIcNAc-R (alphaGal) represent a major immunological hurdle to successful xenotransplantation of pig organs and tissues into humans, and play an important role in providing protective host immunity to various pathogens. However, in spite of the importance of antibodies specific for alphaGal , little is known about how production of these or other anticarbohydrate antibodies is regulated. In the past funding period, we successfully developed a gene therapy based approach to induce B cell tolerance to alphaGal in order to facilitate xenotransplantation, as well as develop a mouse model to study how production of alphaGal specific antibodies is regulated. Using knockout mice, which lack the enzyme that synthesizes alphaGal, and consequently produce aGal reactive natural antibodies, as do humans, we examined whether expression of a retrovirally transduced alphaGT in bone marrow-derived cells could be used to induce tolerance to alphaGal. Expression of alpha(1,3)galactosyltransferase in bone marrow-derived cells of knockout mice completely inhibited production of aGal specific antibodies, resulting in stable long-term tolerance to alphaGal. These data suggest that gene therapy approaches may be used to induce B cell tolerance, effectively reshaping the B cell repertoire. Using gene targeting in embryonic stem cells we also constructed novel immunoglobulin gene knock-in mice in which antibody variable region gene segments encoding aGal specific antibodies were introduced into the immunoglobulin heavy and light chain loci, on either an antigen deficient or antigen sufficient background. In this renewal, we will use these mice to address fundamental issues related to regulation of anti-carbohydrate antibodies. The specific aims are to: 1) Characterize development and regulation of B cells producing aGal specific antibodies; 2) Determine the mechanisms and requirements for anti-carbohydrate B cell tolerance; and 3) Determine the B cell receptor affinity which permits development of B cells that produce antibodies that bind self antigens. These studies should advance our understanding of how production of anti-carbohydrate is regulated, provide insight into how B cells producing antibodies with specificity for self-antigens escape negative selection, and may provide information critical to designing therapeutic agents that could be used to prevent or enhance production of anti-carbohydrate antibodies for clinical applications.